E. coli is the most commonly used host cell for for the mass production of various useful proteins due to its high growth rate and advanced techniques in the fields of fermentology and genetic engineering, available for culturing and genetic engineering of E. coli. A high yield production of recombinant protein requires culturing of the transformed E. coli cells in a high density. To this end, various types of media such as a complex medium, synthetic medium, and semi-synthetic medium, and various methods for feeding media such as a constant-rate feeding, a stepwise increase of the feeding rate, an exponential feeding, or specific growth rate control, pH-stat and DO-stat control, and glucose and acetic acid concentration control have been developed and used to obtain high cell densities of E. coli. But, practically, there is a specific method and optimal condition for culturing each of the transformed strain. Thus for a high yield protein production, it is important to select the most optimal method for feeding media and culturing the cells.
In general, in the production of recombinant protein by recombinant E. coli, the characteristics of corresponding promoter and expressed protein affect the cell growth rate and productivity per each unit of the cells. For the system where protein expression is controlled by a strong promoter, a rapid expression of recombinant protein can disrupt the balance of energy metabolism of the host cell, thereby inhibiting the cell growth. Especially, when the expressed protein has a direct negative effect on host cells, it inhibits the growth of the host cells to greater extent.
For these reasons, protein expression under a strong inducible promoter becomes a significant burden to the host cells. Thus as this burden is minimized, the mass production of recombinant protein can be achieved more easily. Considering the above conditions, optimal expression time may vary depending on the characteristics of the expressed protein even when the same type of expression vector and host cells are used for protein expression. In addition, significance of the effects of expression time on cell growth and expression rates may vary. Furthermore, cell growth rate and cell mass affects a protein production. With the same productivity per cell, use of higher mass of cells can increase the yield of protein production. Therefore, it is important to establish optimal culturing conditions for producing the cell mass with a high density in the production of recombinant proteins by recombinant microorganisms.
Since the protein expression of transformed E. coli is closely related to various intracellular physiological/ecological factors related to the growth environment (stability and number of plasmid copies, transcription and translation efficiency, solubility of expressed proteins, proteolysis, membrane integrity, etc.), it is critical to establish an optimal culturing condition to maximize production yield and productivity. Therefore, in order to express a recombinant protein at high yield, an appropriate inducer such as IPTG needs to be added and media with suitable composition for protein production needs to be fed by appropriate method to the culturing media of recombinant E. coli at high density. If needed, the media composition and method of feeding the same have to be modified for recombinant protein production (Yee and Blanch, 1992, Bio/Technol. 10, 1550-1556).
Methods for expressing a recombinant protein in E. coli can be divided into three representative types. The first method is expression of a recombinant protein as a soluble form in the cytoplasm of the E. coli cell. The second method is expression of a recombinant protein to periplasm of the E. coli cell using a signal sequence. The other method is expression of protein in a form of inclusion body(IB), and this method is used most commonly for expressing a protein at a high yield.
When proteins are expressed in an IB form, the E. coli cells need to be cultured in a high density. In detail, with the similar protein expression rate per each cell, as E. coli cells are grown in higher density, the higher yield of protein products can be obtained. Thus there have been many studies on investigating the method to grow E. coli cells into a high density. Korean Patent No. 10-0235315 discloses a use of fusion protein for over-expression of a human growth hormone, but the cell mass was found to be about 90 to 100 g per liter. Furthermore, it is observed that the final absorvance(OD600) was not higher than 150, in the production process of recombinant protein under the two-step fermentation condition(Microb Cell Fact. 2008 Aug. 7; 7:26). Likewise, a high density-culturing condition for producing protein like salmosin yielded only 65.70 g of IB per liter (J Microbiol Biotechnol. 2011 Oct. 21; (10):1053-6). In addition, culturing condition for producing IFN gamma yielded only about 100 g of cell mass per liter of a cell culture (J Ind Microbiol Biotechnol. 2004 February; 31(2):63-9. Epub 2004 Feb. 19). As a result, there is still a high need for the development of a method of culturing E. coli cells for a higher density.
Based on this background, in an effort to develop a method for producing recombinant protein in a high yield, the present inventors have found that when the host cells are cultured with different types of media between a step of culturing transformed E. coli cells and a step of inducing expression of recombinant protein, a mass production of recombinant protein in the transformed E. coli could be achieved, thereby completing the present invention.